Characterization of DIM-1, an Integron-Encoded Metallo-β-Lactamase from a Pseudomonas stutzeri Clinical Isolate in the Netherlands

Bacterial strains. Pseudomonas stutzeri clinical isolate 13 was identified with the API-20 NE system (bioMérieux, Marcy l'Etoile, France) and confirmed by rRNA gene sequencing. E. coli TOP10 was the host for cloning experiments (19).

Susceptibility testing.Antibiotic-containing disks were used for routine antibiograms by the disk diffusion assay (Sanofi-Diagnostic Pasteur, Marnes-la-Coquette, France), as recommended previously (6). The extended-spectrum β-lactamase (ESBL) double-disk synergy test was performed with disks containing ceftazidime or cefepime and ticarcillin-clavulanic acid on Mueller-Hinton agar plates, and the results were interpreted as described previously (11). MBL detection was performed by using Etest MBL strips (AB Biodisk, Solna, Sweden).

MICs were determined by an agar dilution technique with Mueller-Hinton agar (Sanofi-Diagnostic Pasteur), with an inoculum of 10⁴ CFU per spot, as described previously (16). All plates were incubated at 37°C for 18 h at ambient atmosphere. MICs of β-lactams were determined alone or in combination with a fixed concentration of clavulanic acid (4 μg/ml) or tazobactam (4 μg/ml). MIC results were interpreted according to the guidelines of the CLSI (6).

PCR and hybridization experiments.Total DNA of P. stutzeri 13 was extracted as described previously (2). This DNA was used as a template under standard PCR conditions (25) with a series of primers designed for the detection of the class B β-lactamase genes bla_IMP, bla_VIM, bla_SPM, and bla_SIM (13, 22). Southern hybridizations were performed as described by Sambrook et al. (26), using an enhanced chemiluminescence (ECL) nonradioactive labeling and detection kit (GE Healthcare, Orsay, France).

Cloning experiments, recombinant plasmid analysis, and DNA sequencing.Total DNA of the P. stutzeri 13 isolate was digested with the XbaI restriction enzyme, ligated into the XbaI site of plasmid pBK-CMV, and transformed into E. coli TOP10, as described previously (16). Recombinant plasmids were selected on Trypticase soy agar plates containing amoxicillin (50 μg/ml) and kanamycin (30 μg/ml). The cloned DNA fragments of several recombinant plasmids were sequenced on both strands with an Applied Biosystems sequencer (ABI 3100; Applied Biosystems, Foster City, CA). The entire sequence provided in this study was made of sequences of several plasmids that contained overlapping cloned fragments. The nucleotide and deduced amino acid sequences were analyzed and compared to sequences available at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov ).

Genetic support.Transformation experiments were performed with P. stutzeri 13 DNA and a P. aeruginosa PU21 recipient strain, as described previously (21). Plasmid DNA extraction from P. stutzeri 13 was attempted with a Qiagen Plasmid DNA Maxi kit (Qiagen, Courtaboeuf, France) by the Kieser method (12), and DNA was visualized and sized as described previously (21). Hybridization was performed with a 688-bp probe specific for the bla_DIM-1 gene, generated with internal primers DIM-1A (5′-TCTATTCAGCTTGTCTTCGC-3′) and DIM-1B (5′-TGTTAGAGGCTGTCTCAGCC-3′).

β-Lactamase purification and isoelectric focusing (IEF) analysis.Cultures of E. coli TOP10(pXD-1) were grown overnight at 37°C in 4 liters of Trypticase soy broth containing amoxicillin (100 μg/ml) and kanamycin (30 μg/ml). β-Lactamase was purified by ion-exchange chromatography. Briefly, the β-lactamase extract was obtained by sonication of the cells, resuspended in 100 mM sodium phosphate buffer (pH 7), cleared by ultracentrifugation, treated with DNase, and dialyzed against 20 mM diethanolamine buffer (pH 8.9). This extract was loaded on a Q-Sepharose column, and the β-lactamase-containing fractions were eluted with a linear 0 to 0.5 M NaCl gradient. The fractions containing the highest β-lactamase activity were again dialyzed against the buffer mentioned above, and the procedure was repeated by eluting the protein more slowly, with a linear 0 to 0.2 M NaCl gradient. The purity of the enzyme was estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis. The protein content was measured by the Bio-Rad DC protein assay.

IEF analysis was performed with an Ampholine polyacrylamide gel (pH 3.5 to 9.5) as described previously (15), using a purified β-lactamase extract from a culture of E. coli TOP10(pXD-1). The focused β-lactamases were detected by overlaying the gel with 1 mM nitrocefin (Oxoid, Dardilly, France) in 100 mM phosphate buffer (pH 7.0).

Kinetic measurements.Purified β-lactamase was used for kinetic measurements performed at 30°C with 50 mM HEPES buffer (pH 7.5) supplemented with 50 μM ZnSO₄, using an Ultrospec 2000 UV spectrophotometer (Amersham Pharmacia Biotech), as described previously (3). The specific activity of the purified β-lactamase from E. coli TOP10(pXD-1) was obtained as described previously, with imipenem as the substrate (2). One unit of enzyme activity was defined as the activity which hydrolyzed 1 μmol of imipenem per min per mg of protein. The total protein content was measured with a DC protein assay kit (Bio-Rad, Ivry-sur-Seine, France). In order to evaluate whether the zinc ion concentration might have an impact on DIM-1 hydrolytic activity, specific activities were measured using the purified enzyme and various concentrations of ZnSO₄ (0, 20, 50, 100, 300, and 600 μM and 1 mM), with imipenem as the substrate.

The fifty percent inhibitory concentration (IC₅₀) was determined for DIM-1 as the concentration of EDTA that reduced the hydrolysis rate of 100 μM benzylpenicillin by 50% under conditions in which DIM-1 was preincubated with various concentrations of EDTA for 10 min at 30°C before adding the substrate.

Nucleotide sequence accession number.The nucleotide sequence data reported in this work have been deposited in the GenBank nucleotide database under accession no. GU323019.

Properties of P. stutzeri isolate 13. P. stutzeri isolate 13 was isolated in June 2007 at the VU Medical Center, Amsterdam, Netherlands, from purulent exudate obtained from tibial osteomyelitis in a 55-year-old patient who did not have any history of recent travel or hospitalization elsewhere. P. stutzeri 13 was resistant to ticarcillin, piperacillin, piperacillin-tazobactam, and imipenem, had reduced susceptibility to ceftazidime, cefepime, and cefpirome, and remained fully susceptible to aztreonam. A double-disk synergy test was negative with clavulanate-ceftazidime and clavulanate-imipenem (data not shown) but was positive with the MBL Etest (MIC of IMP of 64 μg/ml versus MIC of IMP-EDTA of 2 μg/ml). This P. stutzeri isolate was also resistant to gentamicin, tobramycin, fluoroquinolones, rifampin, chloramphenicol, and tetracycline and remained susceptible to amikacin, netilmicin, and colistin.

Cloning and sequencing of the β-lactamase gene.Preliminary attempts to detect MBL-encoding genes by PCR failed (data not shown). Using total DNA of P. stutzeri 13 as a template in cloning experiments, several E. coli strains harboring recombinant plasmids, including pXD-1, were obtained. Sequence analysis of a ca. 10-kb cloned fragment of pXD-1 revealed a 756-bp open reading frame (ORF) encoding a 251-amino-acid preprotein corresponding to an Ambler class B β-lactamase designated DIM-1 (for Dutch imipenemase) (1). It possessed the conserved motifs characteristic of MBL enzymes (Fig. 1), including the consensus zinc binding motif HXHXD (residues 116 to 120), together with the critical residues for MBL activity, such as His116, His118, Asp120, His196, Cys221, and His263, according to the BBL nomenclature (9, 31-34) (Fig. 1). According to its amino acid sequence, it can be classified as a member of the subclass 1 MBLs, together with IMP- and VIM-type β-lactamases (25). DIM-1 was distantly related to other Ambler class B β-lactamases. Indeed, the highest percentages of amino acid identity were 52% with GIM-1 (5), 49% with a putative MBL identified in silico in the genome of Shewanella denitrificans (GenBank accession no. NC_007954), and 48% with KHM-1 (27). β-Lactamase DIM-1 shared 45% identity with the widespread IMP-type enzymes and only 30% identity with the VIM-type enzymes. The G+C content of the bla_DIM-1 gene was 43.5%, a value which fits with those of many Gram-negative species but differs significantly from the G+C content of the P. stutzeri genome, which is 63% according to the GenBank database (accession no. NC_009434), thus suggesting horizontal acquisition of bla_DIM-1.

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FIG. 1.

(A) Comparison of the amino acid sequence of the β-lactamase DIM-1 and those of other acquired MBLs (GIM-1, SIM-1, IMP-1, VIM-1, NDM-1, and SPM-1) and several naturally occurring MBLs sharing some degree of similarity (IND-1 from Chryseobacterium indologenes, JOHN-1 from Flavobacterium johnsoniae, SLB-1 from Shewanella livingstonensis, and SFB-1 from Shewanella frigidimarina). Shaded amino acids are those conserved with DIM-1. Numbering of β-lactamases is according to the BBL nomenclature (9). Asterisks indicate conserved amino acid residues, and dashes correspond to the sequence gaps. (B) Secondary structure of DIM-1 compared to that of VIM-2. The beta-strands and alpha-helixes are indicated above the DIM-1 sequence. The conserved residues are indicated in black. The conservative amino acid substitutions are boxed. The figure was obtained with ESPript software (10).

β-Lactam susceptibility.MICs of β-lactams for E. coli TOP10(pXD-1) indicated the expression of an MBL that hydrolyzed expanded-spectrum cephalosporins (including cephamycins) together with carbapenems, that conferred reduced susceptibility to imipenem and meropenem, and that spared aztreonam (Table 1). The addition of β-lactamase inhibitors such as clavulanic acid or tazobactam did not restore the susceptibility to β-lactams.

Biochemical properties of DIM-1.IEF analysis showed that P. stutzeri 13 and E. coli TOP10(pXD-1) had β-lactamase activities with a pI value of 6.2, corresponding to that of DIM-1 (data not shown). The specific activity of the purified β-lactamase DIM-1 was 21 U mg of protein⁻¹ with benzylpenicillin as the substrate. Its overall recovery was 80%, with a 45-fold purification. The purity of the enzyme was estimated to be >95% according to SDS gel electrophoresis analysis (data not shown). The kinetic parameters of DIM-1 showed its broad-spectrum activity against most β-lactams, including oxyimino-cephalosporins, cephamycins, and carbapenems but excluding aztreonam (Table 2). Analysis of the relative hydrolysis rates of DIM-1 showed that cefotaxime was hydrolyzed at a similar level to that of benzylpenicillin, and cefoxitin was also a good substrate. Ceftazidime was significantly hydrolyzed, with a K_m value of 50 μM, reflecting a relatively good affinity of DIM-1 for that substrate, as commonly observed for many MBLs. On the other hand, the monobactam aztreonam was not hydrolyzed by DIM-1, like the case for all MBLs (4, 33). IC₅₀ determinations performed with benzylpenicillin as a substrate showed that DIM-1 activity was inhibited by EDTA (175 μM).

In addition, we observed that the DIM-1 hydrolytic activity was correlated with the zinc ion concentration. Whereas the specific activity of DIM-1 for imipenem was 0.08 U mg of protein⁻¹ in the absence of ZnSO₄, it was 0.12, 0.15, 0.18, 0.28, 0.39, and 0.54 U mg of protein⁻¹ in the presence of increased concentrations of ZnSO₄, i.e., 0, 20, 50, 100, 300, and 600 μM and 1 mM, respectively, thus indicating a significant correlation.

Genetic environment and support of the bla_DIM-1 gene.Sequence analysis of the recombinant plasmid pXD-1 harboring the bla_DIM-1 gene revealed that it was in the form of a gene cassette, which was inserted at the attI1 recombination site (7), similar to the other acquired MBL-encoding genes identified in Gram-negative organisms, such as bla_IMP, bla_VIM, and bla_SIM genes. Analysis of the 5′-end sequence of the integron showed that the gene cassettes' expression was under the control of the P_C promoter, but the secondary promoter (P₂) was not under its active form (14). Thus, the gene cassettes located in that integron are under the control of weak promoter sequences.

The dim-1 gene cassette possessed imperfect core (GTTAGAG) and inverse core (CGCTAAC) sites, with the latter being located inside the bla_DIM-1 coding sequence (23 bp from the 3′ end of the gene). The length of its 59-be sequence was only 31 bp, and the usually conserved 2R and 2L regions of 59-be sites were not detected, indicating that this 59-be was likely truncated and therefore suggesting that the dim-1 cassette may no longer be functional for recombination. A second gene cassette was identified as containing the aadB gene, encoding resistance to aminoglycosides (Fig. 2). The third gene cassette contained the qacH gene, encoding resistance to disinfectants. Inside the qacH gene cassette, the ISKpn4 insertion sequence that targeted the 59-be was identified (24), as previously noticed with other members of the IS1111 family that preferentially target the gene cassette 59-be sites (18, 28).

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FIG. 2.

Schematic map representing the In124 integron structure containing the bla_DIM-1 gene, together with its flanking sequences. The 59-be sites are indicated by white circles. The horizontal arrows indicate the transcription orientations. The insertion sequences ISPst10 and ISKpn4 are represented by rectangles. The IRRs of transposons Tn1721 and Tn3 are represented by black triangles.

Analysis of the right extremity of this integron, named In124, showed that the usually identified 3′-conserved segment made of the qacEΔ1 and sul1 genes was absent, but the tniC gene of transposon Tn5090, encoding a 207-amino-acid-long resolvase, was identified. The tnpA gene of transposon Tn1403 was identified (a Tn3-like transposon), as well as its right inverted repeat (IRR) extremity (Fig. 2).

The left extremity of class 1 integrons, defined by an inverted repeat structure, was identified downstream of the int1 gene, truncating a tnpA gene (lacking 266 bp at its 3′ extremity) that encodes the transposase of transposon Tn1721. The IRR of Tn1721 was also identified and was preceded by a novel insertion sequence element named ISPst10 (Fig. 2). ISPst10 is 1,113 bp long and belongs to the IS30 family, and its transposase shares 90% amino acid identity with that of ISPstI, also identified from a P. stutzeri isolate (http://www-is.biotoul.fr ). Its transposition had generated a 3-bp duplication.

Electrotransformation experiments did not result in the transfer of bla_DIM-1 to either P. aeruginosa PU21 or E. coli TOP10 as a recipient strain. However, analysis of the plasmid content of P. stutzeri 13 identified a single 70-kb plasmid that harbored the bla_DIM-1 gene, as confirmed by Southern hybridization (data not shown). However, the negative mating-out result prevented us from knowing which other antibiotic resistance markers were plasmid associated along with the bla_DIM-1 gene.

In conclusion, we identified a novel MBL sharing weak amino acid identity with other MBLs but sharing similar biochemical properties. The dissemination of the bla_DIM-1 gene among other Gram-negative isolates, especially among the Enterobacteriaceae, remains to be evaluated. Indeed, several pieces of evidence have indicated that environmental species such as Pseudomonas sp. may act as intermediate reservoirs for capturing antibiotic resistance genes from other environmental species and then exchanging those genes with genes of the Enterobacteriaceae.